PROJECT SUMMARY An abnormal chromosome number (aneuploidy) in developing embryos is the leading cause of birth defects and pregnancy loss in women. The majority of aneuploidies are attributed to error-prone meiotic division in oocytes and increase significantly with advanced maternal age. Accurate chromosome segregation is critically dependent on assembly of the microtubule (MT) spindle apparatus and the establishment of correct chromosome-MT interactions. Our studies in mice demonstrate that disruption of meiotic spindle stability can promote chromosome segregation errors, which are not fully resolved -despite spindle checkpoint (SAC) activation. Notably, meiotic spindle formation differs from mitosis as mammalian oocytes (mouse and human) lack typical centrosomes, with centriole loss occurring during fetal stages. Alternatively, spindle MT formation can occur from (i) unique acentriolar microtubule-organizing centers (aMTOCs) and (ii) aMTOC-independent mechanisms in mouse oocytes. In previous studies we identified pericentin (Pcnt) as an essential aMTOC scaffolding protein. Thus, to test aMTOC function we developed a unique oocyte-conditional Pcnt knockdown mouse model using a transgenic RNAi approach. Our recent analysis of oocytes from Tg mice demonstrates that meiotic division is highly error-prone in the absence of maternal Pcnt and disrupted aMTOCs, leading to female subfertility. Importantly, a new study reports that human oocytes (obtained from IVF patients), lack pericentrin and show strikingly similar meiotic errors as our Tg mice. The experiments outlined in this proposal will use this unique genetic model to address the underlying mechanism(s) of spindle formation in oocytes. In Aim 1 we will test the function of key aMTOC-independent mechanisms that promote meiotic spindle formation. We will (i) establish whether Ran activity regulates spindle formation in Pcnt-deficient mouse oocytes and (ii) undertake the first studies to test if the Augmin complex-mediated MT amplification also functions to promote spindle stability in mammalian oocytes. Why human oocytes reportedly lack pericentrin and why aMTOC-independent spindle formation is unstable in oocytes is not known. Thus, experiments in Aim 2 will (i) test the hypothesis that essential aMTOC-associated proteins (pericentrin and ?-tubulin) are disrupted with increasing maternal age. Additionally, we will (ii) determine whether MT dynamics differ in Pcnt-deficient oocytes, leading to spindle instability. Knowledge gained from these studies will provide critical new insight into (i) the key functional mechanisms that promote meiotic spindle formation and (ii) whether disruption of spindle assembly/stability contributes to age-associated aneuploidy in oocytes.